Rapid procedure for the detection of plasmids in Staphylococcus epidermidis

Abstract

A rapid, reproducible, mini-volume assay capable of detecting staphylococcal plasmid DNA in the range of 0.8 to 32 megadaltons has been developed. The assay employs lysostaphin-mediated lysis of cells followed by a short, low-speed centrifugation and does not require treatment with ribonuclease or protease or deproteinization with phenol. A period of only 24 h may be required to detect the presence and size of a plasmid once an organism has been isolated. This method has been used to study the plasmid ecology of Staphylococcus epidermidis and to correlate the presence or absence of plasmids with tetracycline, chloramphenicol, neomycin, penicillin, and cadmium resistances. Simple agarose gel electrophoretic procedures for the detection and characterization of plasmid DNA in gram-negative bacteria have been published previously (2, 5, 14). All of these procedures utilize detergent-mediated lysis, which is ineffective against gram-positive bacteria without prior treatment with lysozyme or lysostaphin in the case of staphylococci. We have developed a fast, reproducible, small-volume assay capable of detecting staphylococcal plasmid DNA in the range of 0.8 to 32 megadaltons. This assay is a modification of the Brij-lysostaphin cleared-lysate procedure commonly used for the isolation of plasmid DNA in Staphylococcus aureus (13). This procedure has been used in our laboratory to detect plasmid-free strains of Staphylococcus epidermidis for use as recipients in transduction and transformation experiments and to correlate antibiotic resistances with the presence or absence of plasmids of size classes typically coding for these resistances in staphylococci (4). MATERIALS AND METHODS Bacterial strains and media. Strains of S. aureus and S. epidermidis used in this study and their resistance patterns are shown in Table 1. Most strains were obtained from nasal cultures of young adults. Grampositive cocci were designated as staphylococci if they were catalase positive and produced acid from glucose

Characterization and construction of molecular cloning vehicles within Staphylococcus aureus

“…Phage typing and antibiotic resistance patterns of transformants were determined and compared with that of the recipient strain to detect contaminants. In addition, the plasmid DNA content of the donor strain and transformant were compared by the mini-volume Brij-cleared lysate procedure (36).…”

“…Mini-volume Brij-cleared lysate procedure. The mini-volume Brij-cleared lysate procedure was as described by Wilson et al (36).…”

Diversity of plasmids inStaphylococcus saprophyticusisolated from urinary tract infections in women

“…Plasmid DXA was isolated by a technique adapted from modifications of the methods of Wilson. Totten & Baldwin (1978) and of Kado & Liu (1981).…”